DESCRIPTION: Enamel fluorosis is a defect in enamel development that occurs after exposure to excess fluoride. Fluorotic enamel is more porous, and contains more proteins than sound enamel. The mechanisms by which fluoride alters enamel formation remain not well understood. In this competing renewal, we propose studies to begin to explore the indirect effects of fluoride resulting from changes in the forming enamel matrix, secondary to fluoride incorporation in the growth of enamel crystals. We will also continue to explore the direct effects of fluoride on the cells, matrix proteins, and proteinases that form enamel. We hypothesize that both of these effects of fluoride on enamel formation are mechanisms by which fluoride alters enamel formation, and propose the following Specific Aims: 1). To determine the indirect effects of fluoride on enamel formation and biomineralization, including acidification of the enamel space that indirectly affects cell and matrix function. 2). To determine how fluoride interacts with enamel proteins including amelogenins and proteinases resulting in changes of amelogenin processing and crystal growth. 3) To determine how fluoride directly affects ameloblast differentiation, including altered expression of matrix proteins and proteinases, and apoptosis. The changes in pH resulting from fluoride exposure will be measured, and the effect of amelogenin and bicarbonate buffering in the enamel matrix and in the ameloblasts will be determined in amelogenin knockout mice and HCO37C1 (Ae2) exchange knockout mice. The direct interactions of fluoride with amelogenins and matrix proteinases, and the effect on crystal growth will be measured in an in vitro crystal growth system. We will use ameloblast cell culture to identify fluoride related changes in gene expression related to cell proliferation, apoptosis, and measure changes in activity of matrix enzymes that are related to mineralization. These studies are important to understand how fluorosis forms in enamel. By understanding the mechanisms by which fluorosis occurs, we will be able to identify individuals and conditions which may be more sensitive to or affected by fluoride, and will be better able to prevent the formation of enamel fluorosis.